Galectin-3 and prohibitin 1 are autoantigens in IgG4-related cholangitis without clear-cut protective effects against toxic bile acids

Remco Kersten¹ David C. Trampert¹ Lowiek M. Hubers¹ Dagmar Tolenaars¹ Harmjan R. Vos² Stan van De Graaf¹ Ulrich Beuers^1*

• ¹Amsterdam University Medical Centers, Department of Gastroenterology & Hepatology, Tytgat Institute for Liver and Intestinal Research, Netherlands
• ²Oncode Institute and Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, Netherlands, Netherlands

Background & Aims: IgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease, a systemic B-cell-driven fibroinflammatory disorder. Four autoantigens have recently been described in IgG4-RD; annexin A11, galectin-3, laminin 511-E8, and prohibitin 1. We have previously reported a protective role of annexin A11 and laminin 511-E8 in human cholangiocytes against toxic bile acids. Here we explored a potentially protective role of the carbohydrate-binding lectin galectin-3 and the scaffold proteins prohibitin 1 and 2.
Methods: Anti-galectin-3, anti-prohibitin 1 and 2 autoantibody positivity in IRC and healthy and disease (primary sclerosing cholangitis, PSC) control sera was assessed by ELISA/LC-MS. Human H69 cholangiocytes were subjected to shRNA knockdown targeting galectin-3 (LGALS3), prohibitin 1 (PHB1) and prohibitin 2 (PHB2). H69 cholangiocytes were also exposed to recombinant galectin-3, the inhibitor GB1107, and to recombinant prohibitin 1 and the pan-prohibitin inhibitor rocaglamide. Protection against bile acid toxicity was assessed by intracellular pH (pHi) measurements using BCECF-AM; 22,23-3H-glycochenodeoxycholic acid (3H-GCDC)-influx; and GCDC-induced apoptosis using Caspase-3/7 assays.
Results: Anti-galectin-3 autoantibodies were detected in 13.5% of individuals with IRC, but not PSC. Knockdown of LGALS3 and galectin-3 inhibition with GB1107 did not affect pHi, whereas recombinant galectin-3 incubation lowered pHi. LGALS3 knockdown increased GCDC-influx, but not GCDC-induced apoptosis. GB1107 reduced GCDC-influx and GCDC-induced apoptosis. Recombinant galectin-3 tended to decrease GCDC-influx and GCDC-induced apoptosis. Anti-prohibitin 1 autoantibodies were detected in 61.5% and 35.7% of individuals with IRC and PSC, respectively. Knockdown of PHB1, combined PHB1/2 KD, treatment with rocaglamide and recombinant prohibitin 1 all lowered pHi. Knockdown of PHB1, PHB2 or combined PHB1/2 did not alter GCDC-influx, yet knockdown of PHB1 increased GCDC-induced apoptosis. Conversely, rocaglamide reduced GCDC-influx but did not attenuate GCDC-induced apoptosis. Recombinant prohibitin 1 did not affect GCDC-influx or GCDC-induced apoptosis. Finally, anti-galectin-3 and anti-prohibitin 1 autoantibody pretreatment did not lead to increased GCDC-influx.
Conclusions: A subset of individuals with IRC have autoantibodies against galectin-3 and prohibitin 1. Gene-specific knockdown, pharmacological inhibition and recombinant protein substitution did not clearly disclose a protective role of these autoantigens in human cholangiocytes against toxic bile acids. Involvement of these autoantibodies in processes surpassing epithelial secretion remains to be elucidated.